Process for producing a cyclic ether

ABSTRACT

Described is a microbiological method for producing the cyclic ether having the chemical structure: ##STR1## using a sclareol derivative having one of the structures: ##STR2## and using the microorganism: Cryptococcus laurentii, ATCC 20920.

PRIOR RELEVANT APPLICATIONS

This application is a continuation-in-part of application for U.S. Pat.Ser. No. 399,826 filed on Aug. 28, 1989 now U.S. Pat. No. 4,970,163issued on Nov. 13, 1990.

BACKGROUND OF THE INVENTION

The compound sclareolide having the structure: ##STR3## has been foundto be a valuable intermediate in preparing the compound having thestructure: ##STR4## an important material for use in perfumery.

The compound which is the cyclic ether having the structure: ##STR5##has the been shown to be useful in U.S. Pat. No. 4,798,799 issued Jan.17, 1989 as an intermediate in the creation of the compound having thestructure: ##STR6## The compound having the structure: ##STR7## has beenshown to be useful in U.S. Pat. No. 4,798,799 issued Jan. 17, 1989 as anintermediate in the creation of the compound having the structure:##STR8## and has also been shown to be a useful precursor of thecompound having the structure: at column 8, lines 58-60 of U.S. Pat. No.4,798,799.

Indeed, U.S. Pat. No. 4,798,799 discloses the utilization of a culturecontaining the microorganism Hyphozyma roseoniger having the identifyingcharacteristics of CBS 214.83 and ATCC 20604 capable of producing thediol having the structure: ##STR9## in a recoverable quantity upon thetransformation of compounds including the compound having the structure:##STR10## (sclareol) as well as the cyclic ether having the structure:##STR11##

Table IV, thereof at column 12, lines 15-28 discloses yield of 96% whencarrying out the reaction: ##STR12## under fermentation conditions usingATCC 20624.

There is no teaching or suggestion in the prior art of either (a)carrying out the reaction: ##STR13## via microbiological methods wherebythe compound having the structure: ##STR14## is formed in relativelyhigh yields or (b) carrying out the reaction: ##STR15## via amicrobiological method using the organism Bensingtonia ciliata, ATCC20919 or (c) carrying out the reaction: ##STR16## via a microbiologicalmethod using the organism Cryptococcus laurentii, ATCC 20920.

Furthermore, the organisms:

Cryptococcus albidus, ATCC 20918;

Bensingtonia ciliata, ATCC 20919;

Cryptococcus laurentii, ATCC 20920; and

Cryptococcus albidus, ATCC 20921.

are novel organisms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a GC-MS spectrum for the starting material of Example III. Thepeak indicated by reference numeral 21 is the peak for sclareol havingthe structure: ##STR17## The peak indicated by reference numeral 20 isthe peak for the internal standard, the compound having the structure:##STR18##

FIG. 2 is the NMR spectrum for the reaction product of Example II, thecompound having the structure: ##STR19##

FIG. 3 is the NMR spectrum for the reaction product of Example IIIhaving the structure: ##STR20##

FIG. 4 is the NMR spectrum for the reaction product of Example IX, thecompound having the structure: ##STR21##

SUMMARY OF THE INVENTION

The present invention concerns biologically pure cultures of themicroorganisms:

Cryptococcus albidus, ATCC 20918;

Bensingtonia ciliata, ATCC 20919;

Cryptococcus laurentii, ATCC 20920; and

Cryptococcus albidus, ATCC 20921.

In another embodiment the present invention concerns cultures containingthe microorganisms:

Cryptococcus albidus, ATCC 20918;

Bensingtonia ciliata, ATCC 20919;

Cryptococcus laurentii, ATCC 20920; and

Cryptococcus albidus, ATCC 20921,

said cultures individually capable of producing either the diol havingthe structure: ##STR22## or the cyclic ether having the structure:##STR23## or sclareolide having the structure: ##STR24## as follows:Cryptococcus albidus, ATCC 20918 and

Cryptococcus albidus, ATCC 20921,

capable of producing sclareolide having the structure: ##STR25## from amixture of sclareol having the structure: ##STR26## and episclareolhaving the structure: ##STR27## Bensingtonia ciliata, ATCC 20919 capableof producing the diol having the structure: ##STR28## from a mixture ofsclareol having the structure: ##STR29## and episclareol having thestructure: ##STR30## and Cryptococcus albidus ATCC 20920 capable ofproducing the cyclic either having the structure: ##STR31## from amixture of sclareol having the structure: ##STR32## and episclareolhaving the structure: ##STR33## under aerobic conditions in an aqueousnutrient medium.

In still another embodiment the present invention concerns mixturesprepared by cultivating the microorganisms (individually) as follows:

Cryptococcus albidus having the identifying characteristics of ATCC20918;

Bensingtonia ciliata having the identifying characteristics of ATCC20919;

Cryptococcus laurentii having the identifying characteristics of ATCC20920; and

Cryptococcus albidus having the identifying characteristics of ATCC20921

under aerobic conditions in an aqueous nutrient medium.

A further embodiment of the present invention concerns a process forpreparing sclareolide having the structure: ##STR34## which comprisescultivating either the organism Cryptococcus albidus having theidentifying characteristics of ATCC 20918 or Cryptococcus albidus havingthe identifying characteristics of ATCC 20921 under aerobic conditionsin an aqueous nutrient medium containing one or more of the compoundshaving the structures: ##STR35##

Still a further embodiment of the present invention concerns a processfor preparing the diol having the structure: ##STR36## which comprisescultivating the microorganism Bensingtonia ciliata having theidentifying characteristics of ATCC 20919 under aerobic conditions in anaqueous nutrient medium containing one or more of the compounds selectedfrom the group consisting of:

(i) sclareol having the structure: ##STR37## (ii) episclareol having thestructure: ##STR38## (iii) the acetate having the structure: ##STR39##

A further embodiment of the present invention concerns a process forpreparing the cyclic ether having the structure: ##STR40## whichcomprises cultivating the microorganism Cryptococcus laurentii havingthe identifying characteristics of ATCC 20920 under aerobic conditionsin an aqueous nutrient medium containing one or more of the compound'sselected from the group consisting of:

(i) Sclareol having the structure: ##STR41## (ii) episclareol having thestructure: ##STR42## (iii) the acetate having the structure: ##STR43##

The transformation processes involve cultivation of one of themicroorganisms:

Cryptococcus albidus, ATCC 20918;

Bensingtonia ciliata, ATCC 20919;

Cryptococcus laurentii, ATCC 20920; or

Cryptococcus albidus, ATCC 20921

in an aqueous nutrient medium in the presence of one, two or all of thecompounds having the structures: ##STR44## Thus, these compounds may beused singularly or as a mixture containing any number of said compounds.

Thus, in carrying out the reaction using:

Cryptococcus albidus, ATCC 20918 or

Cryptococcus albidus, ATCC 20921

the following reactions can take place: ##STR45##

In carrying out the reaction using Bensingtonia ciliata, ATCC 20919 thefollowing reactions can take place: ##STR46##

In carrying out the reaction using Cryptococcus laurentii ATCC 20920,the following reactions can take place: ##STR47##

The form in which the microorganisms are used is not critical. They canbe used as the culture (suspension), i.e., including the cells and thecorresponding nutrient solution, or in the form of cells suspended in abuffer solution. The cells, or an enzyme extract thereof, may beimmobilized on a suitable solid support, which may then be used toeffect transformations.

The suspended culture mixture is prepared by inoculation of a suitableaqueous nutrient medium with the microorganisms. A suitable nutrientmedium is one which contains nitrogen sources, inorganic salts, growthfactors, the desired substrate(s), and optionally other carbon sources.Some carbon sources suitable for use in the inventive process include,for example, glucose, galactose, L-sorbose, maltose, sucrose,cellosbiose, trehalose, L-arabinose, L-rhamnose, ethanol, glycerol,L-erythrithol, D-mannitol, lactose, melibiose, raffinose, melezitose,starch, D-xylose, D-sorbitol, a methyl-D-glucoside, lactic acid, citricacid and succinic acid. Suitable nitrogen sources include, for example,nitrogen-containing organic substances such as peptone, meat extract,yeast extract, corn steep liquor, casein, urea, amino acids, ornitrogen-containing inorganic compounds such as nitrates, nitrites andinorganic ammonium salts. Suitable inorganic salts include, for example,phosphates of magnesium, potassium, calcium, or sodium. The abovementioned culture medium nutrients may be supplemented with, forexample, one or more vitamins of the B group and/or one or more traceminerals such as Fe, Mo, Cu, Mn, and B, as desired. The vitamins ortrace minerals are not necessary when a small amount of yeast extract isadded to the medium. Addition of an antibiotic, such as chloroamphenicolor chlorotetracycline, may be desirable when bacterial contamination isa problem. The cultivation of the microorganism may be carried out as astationary culture or as a submerged (e.g., shaking culture, fermentorculture) under aerobic conditions. One may suitably work in the pH rangeof from about 2.5 to about 9.0, and preferably in the range of fromabout 3.0 to about 7.5 and most preferably between about 3.0 and 6.5.The pH may be regulated by the addition of inorganic or organic acids,such as hydrochloric acid, acetic acid, and oxalic acid, or by theaddition of bases, such as sodium hydroxide, and ammonium hydroxide, orby the addition of a buffer, such as phosphate or phthalate. Theincubation temperature should suitably be maintained between about 12°C. and about 33° C., with a range between about 15° C. and about 30° C.being more preferred, and a range between about 18° C. and about 28° C.being most preferred.

The process in accordance with this invention may be convenientlycarried out by adding one or a mixture of the compounds having thestructures: ##STR48## to the nutrient medium at the onset ofcultivation, as the sole carbon source. Alternatively, the substrate maybe added in combination with another carbon source, such as dextrose,either during cultivation, or when the carbon source is depleted. Theonly restriction on the concentration of substrate in the culture mediumis that of being able to effectively aerate the culture. However, thesubstrate concentration is preferably in the range of between about 0.1g/L and about 130 g/L, more preferably in the range of between about 0.5g/L and about 120 g/L, and most preferably in the range between about2.5 g/L up to about 100 g/L. The transformation can be suitably carriedout under any of the above mentioned conditions.

The total transformation time (after initial cultivation period) mayvary depending on the composition of the nutrient medium and thesubstrate concentration. In general, shaking flask cultures require formbetween about 12 hours and about 264 hours. However, when a fermentor isused the cultivation time may be reduced to about 48 hours or less.

The transformation may be carried out using the cells of themicroorganism isolated from the culture solution, or with an enzymeextract isolated from the cells in a manner well known to the art. Inthis case, the transformation can be conveniently carried out in avariety of aqueous nutrient media including, for example, in a buffersolution, in a physiological salt solution, in a fresh nutrientsolution, or in water. The isolated cells or enzyme extract may beimmobilized on a solid support and the desired transformation effected.Also, transformation of the substrate may be effected by mutants of thisorganism. Such mutants can be readily obtained by method well known inthe art, for example, by exposing the cells to UV or X-rays, or knownmutagenic substances, such as, for example, acridine orange.

The substrate can be added to the medium as a powder, or a slurry in anemulsifier such as TWEEN® 80 (polyoxyethylenesorbitan mono-oleate), oras a solution in an emulsifier, or as a solution in a hydrophilicsolvent such as acetone, methanol, ethanol, ethylene glycol, or dioxan.A surface-active agent, or a dispersion agent can also be added to anaqueous suspension of the substrate, or the substrate can be emulsifiedusing ultrasound.

Conventional antifoam agents, such as silicone oils (e.g., UCON),polyalkyleneglycol derivatives, maize oil, or soya oil, can be used tocontrol foaming.

The transformation of the substrate can be monitored using standardanalytical techniques such as GLC, TLC, HPLC, IR and NMR. If a rapiddisappearance of the substrate is observed more substrate can then beadded, in order to maximize the transformation capacity of themicroorganism. The process is generally terminated when most of thesubstrate has disappeared from the culture medium. Depending upon themicroorganism used, the compound having the structure: ##STR49## or thecompound having the structure: ##STR50##

or the compound having the structure: ##STR51## may be recovered fromthe aqueous nutrient medium.

The compound having the structure: ##STR52## may be reacted to form thecompound having the structure: ##STR53## according to the reaction:##STR54## The compound having the structure: ##STR55## may be cyclizedto the compound having the structure: ##STR56## as stated at lines 52and 53, at column 8 of U.S. Pat. No. 4,798,799 the specification forwhich is incorporated by reference herein. The compound having thestructure: ##STR57## may also be used as is for its flavor or fragrancevalue or it may be reduced to the compound having the structure:##STR58## according to the reaction: ##STR59##

Production of the compound having the structure: ##STR60## may takeplace by (i) first reducing the compound having the structure: ##STR61##to the diol having the structure: ##STR62## or (ii) first reacting thecyclic ether having the structure: ##STR63## via fermentation to producethe diol having the structure: ##STR64## and then rearranging the diolto form the compound having the structure: ##STR65## according to thereaction: ##STR66## or fermenting the compound having the structure:##STR67## according to the reactions: ##STR68##

Preferably, the reduction reaction, to wit: ##STR69## is carried out inthe presence of an inert solvent such as toluene using a reducing agentsuch as VITRIDE®, registered trademark of Eastman Kodak Company ofRochester, N.Y. identifying sodium bis (2-methoxyethoxy) aluminumhydride specifically described in U.S. Pat. No. 3,507,895 thespecification for which is incorporated herein by reference. Thereaction is preferably carried out at a temperature in the range of fromabout 70° C. up to about 90° C. for a period of between about one hourand about three hours.

The rearrangement reaction, to wit: ##STR70## is preferably carried outin basic media, e.g., aqueous alkali metal hydroxide such as aqueouspotassium hydroxide or aqueous sodium hydroxide as more specificallydescribed in Example VIII, infra.

Isolation and purification of the compounds having the structures:##STR71## from the fermentation broths may be achieved by conventionaltechniques including, filtration or centrifugation, solvent extraction,distillation, crystallization, and the like.

The compound having the structure: ##STR72## may be converted to thecompound having the structure: ##STR73## by conventional cyclizationmethods well known in the art as specified at lines 58-68, at column 8of U.S. Pat. No. 4,798,799 and at column 9, lines 1 and 2 of U.S. Pat.No. 4,798,799.

Each of the microorganisms employed in this invention was isolated froma soil sample obtained from various geographical locations. Each of thestrains has been deposited with the American Type Culture Collectionwith the accession numbers as follows:

Cryptococcus albidus, ATCC 20918;

Bensingtonia ciliata, ATCC 20919;

Cryptococcus laurentii, ATCC 20920; and

Cryptococcus albidus, ATCC 20921.

The organisms Bensingtonia ciliata and Cryptococcus laurentii were alsostudied by Centralbureau voor Schimmel Cultures (CBS). CBS assigned bothof these organisms the name:

Lecythophere hoffmannii (van Beijma), W. Gams (synonym Phialophorahoffmannii)

because this is a filamentous fungus according to CBS.

The organisms Cryptococcus albidus, ATCC 20918 was also studied byCentraalbureau voor Schimmelkultur (CBS). CBS named this culture to beCryptococcus albidus var. albidus (Saito) Skinner.

the organism Cryptococcus albidus, ATCC 20918 is described as follows:

Morphology: Growth in liquid medium showed unipolar budding cells. Afilm stayed on the surface of the liquid while heavy sediment wasobserved. Growth on solid agar was unicellular with colonies being whileto slightly pinkish, very shiny, bright, slimy, round and with smoothborders. No pseudohyphae were formed on corn meal aga.

Physiology and Biochemistry:

    ______________________________________                                                        Carbon Assimilation:                                                          (Growth)                                                      ______________________________________                                        Carbon Assimilation:                                                          (Growth)                                                                      Glucose        +      D-ribose        U                                       Galactose      +      L-rhamnose      +                                       L-sorbose      +      D-glucosamine   +                                       Maltose        +      Ethanol         U                                       Sucrose        +      Erythritol      -                                       Cellobiose     +      Glycerol        U                                       Trehalose      +      Adonitol (Ribitol)                                                                            +                                       Lactose        +      Dulcitol (Galactitol)                                                                         +                                       Melibiose      -      D-mannitol      +                                       Raffinose      +      D-sorbitol (glucitol)                                                                         +                                       Melezitose     +      a-methyl-D-glucoside                                                                          +                                       Inulin         -      Salicin         +                                       Soluble Starch -      Inositol        +                                       D-xylose       -      Lactic acid     -                                       L-arabinose    -      Citric acid     -                                       D-arabinose    -                                                              Succinic acid  +                                                              Growth at 30° C.                                                                      +                                                              Growth at 37° C.                                                                      -                                                              Vitamin free growth                                                                          -                                                              Splitting of arbutin                                                                         +                                                              Nitrogen assimilation:                                                        NH.sub.4 NO.sub.3                                                                            +                                                              KNO.sub.3      +                                                              NO.sub.2       +                                                              Ethylamine     +                                                              Fermentation (Acid from):                                                     Glucose        -                                                              Galactose      -                                                              Maltose        -                                                              Sucrose        -                                                              Lactose        -                                                              Raffinose      -                                                              Melibiose      -                                                              Inulin         -                                                              Cellobiose     -                                                              Melezitose     -                                                              Starch         -                                                              Trehalose      -                                                              ______________________________________                                         Note: U = undecided or questionable                                      

The organism Bensingtonia ciliata, ATCC 20919 is described as follows:

Morphology: On Yeast Maintenance Broth (ATCC medium #200) cells areglobose with 1-3 buds per cell. On solid medium cells become filamentouswith ballistospores produced. Colonies are tan-salmon in color, flat,dull, with smooth edges. On Corn Meal Agar (ATCC medium #307) truemycelium was noticed after 3 weeks.

Physiology:

    ______________________________________                                                         Carbon                                                                        Assimilation:                                                ______________________________________                                        Carbon Assimilation:                                                          Glucose        +       D-ribose       -                                       Galactose      +       L-rhamnose     +                                       L-sorbose      +       D-glucosamine  +                                       Maltose        +       Ethanol        W                                       Sucrose        +       Erythritol     +                                       Cellobiose     +       Glycerol       +                                       Trehalose      +       Adonitol (Ribitol)                                                                           W                                       Lactose        -       Dulcitol (Galactitol)                                                                        W                                       Melibiose      +       D-mannitol     W                                       Raffinose      +       D-sorbitol (glucitol)                                                                        W                                       Melezitose     +       a-methyl-D-glucoside                                                                         W                                       Inulin         -       Salicin        W                                       Soluble Starch +       Inositol       -                                       D-xylose       +       Lactic acid    -                                       L-arabinose    +       Citric acid    -                                       D-arabinose    -       Succinic acid  W                                       Vitamin free growth                                                                          -                                                              Nitrogen assimiliation:                                                       NH.sub.4 NO.sub.3                                                                            W                                                              Growth at elevated temp:                                                      30° C.  W/-                                                            37° C.  -                                                              Bensintonia ciliata,                                                          ATCC 20919 (Cont'd.)                                                          Taxonomic Description:                                                        Bensingtonia ciliata                                                          C.T. Ingold                                                                   Hyphomycete                                                                   (Fungi Imperfecti)                                                            ______________________________________                                         Note: W = weak                                                           

A ballistosporic fungus (Spores forcibly abjected).

The ballistospores are colorless, ovoid, 2×5 c, mostly 8×5 c in liquidculture, pointed at the apex and with a flattened base.

The ballistospores germinate with the formation of yeast-likeblastospores that upon repeated spore formation result in typical yeastcolonies (see photo).

Some ballistospores germinate with the formation of short hyphae thatproduce ballistospores and with repeated spore discharge result incolonies entirely of the hyphae type (see photo).

Evaluation from the following media:

    ______________________________________                                        ATCC medium                                                                   ______________________________________                                        #307          Corn Meal Agar (Difco 0386) and                                               1/2 strength Corn Meal Agar                                     #200          Yeast Malt Agar (Difco 0712)                                    #331          Neuropora Agar (Difco 0321)                                     #1245         YEPD                                                            #324          Malt Extract Agar (Difco 0024)                                  #336          Potato Dextrose Agar                                            #343          V-8 Juice Agar                                                  ______________________________________                                    

REFERENCES

1. Ingold, C. T. (1986) Bensingtonia ciliata Gen. et. sp. nov.,Ballistoporic Fungus. Trans. Br. Mycol. Soc. 86(2): 325-328.

2. Ingold. C. T. (1988) Further Observations on Bensingtonia ciliata.Trans. Br. Mycol. Soc. 91(1): 162-166.

Cryptococcus laurentii, ATCC 20920 is described as follows:

Physiology and Biochemistry:

    ______________________________________                                        Carbon Assimilation:                                                          (Growth)                                                                      Glucose      +        D-ribose      + weak                                    Galactose    +        L-rhamnose    +                                         L-sorbose    + weak   D-glucosamine V                                         Maltose      +        Ethanol       +                                         Sucrose      +        Erythritol    +                                         Cellobiose   +        Glycerol      +                                         Trehalose    +        Adonitol (Ribitol)                                                                          + weak                                    Lactose      + weak   Dulcitol      V                                                               (Galactitol)                                            Melibiose    +        D-mannitol    +                                         Raffinose    +        D-sorbitol (glucitol)                                                                       +                                         Melezitose   +        a-methyl-D-   +                                                               glucoside                                               Inulin       +        Salicin       +                                         Soluble Starch                                                                             +        Inositol      +                                         D-xylose     +        Lactic acid   +                                         L-arabinose  +        Citric acid   +                                         D-arabinose  + weak   Succinic acid +                                         Nitrogen Assimilation:                                                        NH.sub.4 NO.sub.3                                                                          +                                                                KNO.sub.3    +                                                                NO.sub.2     +                                                                Ethylamine   +                                                                Vitamin free growth                                                                        +                                                                Splitting of arbutin                                                                       +                                                                Cryptococcus laurentii,                                                       ATCC 20920 (Cont'd.)                                                          Fermentation                                                                  (Gas Production):                                                             Glucose      -                                                                Galactose    -                                                                Maltose      -                                                                Sucrose      -                                                                Lactose      -                                                                Raffinose    -                                                                Melibiose    -                                                                Inulin       -                                                                Cellobiose   -                                                                Melezitose   -                                                                Starch       -                                                                Trehalose    -                                                                ______________________________________                                         Note: V = variable.                                                      

Morphology: Pink slimy colonies; round budding cells, heavy sediment inflask; no Dalmau plate thin hyphae were formed.

Reference: C. P. Kurtzman, Mycologia 65; p. 388-395, 1973.

Cryptococcus albidus, ATCC 20921 is described as follows:

Physiology and Biochemistry:

    ______________________________________                                        Carbon Assimilation:                                                          ______________________________________                                        Carbon Assimilation:                                                          Growth                                                                        Glucose        +      D-ribose        -                                       Galactose      +      L-rhamnose      +                                       L-sorbose      +      D-glucosamine   +                                       Maltose        +      Ethanol         V                                       Sucrose        +      Erythritol      -                                       Cellobiose     +      Glycerol        V                                       Trehalose      +      Adonitol (Ribitol)                                                                            V                                       Lactose        +      Dulcitol (Galactitol)                                                                         +                                       Melibiose      -      D-mannitol      +                                       Raffinose      +      D-sorbitol (glucitol)                                                                         +                                       Melezitose     +      a-methyl-D-glucoside                                                                          +                                       Inulin         -      Salicin         +                                       Soluble Starch +      Inositol        +                                       D-xylose       +      Lactic acid     -                                       L-arabinose    +      Citric acid     -                                       D-arabinose    +      Succinic acid   +                                       Nitrogen Assimilation:                                                        NH.sub.4 NO.sub.3                                                                            +                                                              KNO.sub.3      +                                                              NO.sub.2       +                                                              Ethylamine     +                                                              Vitamin free growth                                                                          +                                                              Splitting of arbutin                                                                         +                                                              Cryptococcus albidus,                                                         ATCC 20921 (Cont'd)                                                           Fermentation                                                                  (Gas Production):                                                             Glucose        -                                                              Galactose      -                                                              Maltose        -                                                              Sucrose        -                                                              Lactose        -                                                              Raffinose      -                                                              Melibiose      -                                                              Inulin         -                                                              Cellobiose     -                                                              Melezitose     -                                                              Starch         -                                                              Trehalose      -                                                              ______________________________________                                         Note: V =  variable.                                                     

Morphology: Yellowish-tan, slimy colonies; round budding cells; heavysediment in the flask, no pseudomycelium or true mycelium.

The following examples serve to illustrate embodiments of the inventionas it is now preferred to be practiced but in no way are said Examplesmeant to limit the scope thereof. Unless otherwise stated weights are ingrams, temperatures are in degrees centigrade and pressure is in mm/Hg.

EXAMPLE I Effect of pH on Conversion of Sclareol to Sclareolide UsingCryptococcus Albidus (Saito [Skinner Var. Albidus])(ATCC 20918)Reactions ##STR74##

The following medium was prepared:

    ______________________________________                                               NH.sub.4 NO.sub.3                                                                            0.1%                                                           KH.sub.2 PO.sub.4                                                                            0.1%                                                           MgSO.sub.4.7H.sub.2 O                                                                        0.05%                                                          Yeast Extract  0.2%                                                    ______________________________________                                    

Eleven 500 ml flasks each containing 100 ml of medium and 1 g ofsclareol in TWEEN® 80 (ratio of sclareol of TWEEN® 80=2:1).

Each flask was inoculated with 5 ml of a 24 hour culture grown ondextrose at 25° C. and 150 rpm. Product and substrate were monitored byTLC against a known standard.

The "substrate" is sclareol which is an 80:20 mixture of the compoundhaving the structure: ##STR75## and the compound having the structure:##STR76## the "Intermediate" is the compound having the structure:##STR77## The "product" is sclareolide, the compound having thestructure: ##STR78##

                  TABLE I                                                         ______________________________________                                        Flask         DURATION                                                        No.    pH     24 Hours    48 Hours  72 Hours                                  ______________________________________                                        1      2.5    TP + S + TI P + S + I P + S + TI                                2      3.0    P + TS + I  P + S + I P                                         3      3.5    P + TS + TI P + TI    P                                         4      4.0    P + TS + I  P + TI    P + TI                                    5      4.5    P + TS + I  P + I     P + TI                                    6      5.0    P + TS + I  P + I     P + TI                                    7      7.0    P + S + I   P + I     P + I                                     8      7.5    P + S + I   P + I     P + I                                     9      8.0    P + S + I   P + TI + I                                                                              P + I                                     10     8.5    P + S + I   P +  TS + I                                                                             P + I                                     11     9.0    P + S + I   P + TS + I                                                                              P + I                                     ______________________________________                                         TS: Trace Substrate                                                           S: Sustrate                                                                   TP: Trace Product                                                             P: Product                                                                    I: Intermediate                                                               TI: Trace Intermediate                                                   

EXAMPLE II Preparation of Diol Intermediate Reactions ##STR79##

During screening of ten soil samples from Greenwood Forest, BarnegatTownship, N.J., several flasks showed a spot on the TLC corresponding tothe compound having the structure: ##STR80## The following medium wasprepared:

    ______________________________________                                               NH.sub.4 NO.sub.3                                                                            0.2%                                                           KH.sub.2 PO.sub.4                                                                            0.1%                                                           MgSO.sub.4.7H.sub.2 O                                                                        0.05%                                                          Yeast Extract  0.2%                                                           Dextrose       1.0%                                                    ______________________________________                                    

Into a 500 ml flask was placed 10 ml medium and 1.0 g of a 50:50 mixtureof sclareol powder:TWEEN® 80. The flask was inoculated with 400microliters of isolate of Bensingtonia ciliata, ATCC 20919. After oneweek at 25° C. and 150 rpm, the resulting product was extracted with 330ml of ethyl acetate and the extract dried over anhydrous sodium sulfate.The solvent was removed on a rotary evaporator. The residue wasdissolved in hot hexane and ethyl acetate. The resulting extract waspermitted to evaporate for a period of 24 hours whereupon were obtainedpure crystals (350 mg) of the compound having the structure: ##STR81##were recovered.

FIG. 2 is the NMR spectrum of the compound having the structure:##STR82##

EXAMPLE III Preparation of Sclareolide Using Cryptococcus albidus (Saito[Skinner Var. Albidus]), ATCC 20918 Reactions ##STR83##

    ______________________________________                                        MEDIUM         FERMENTER PARAMETERS                                           ______________________________________                                        NH.sub.4 NO.sub.3                                                                        0.2%    Temperature:    25° C.                              KH.sub.2 PO.sub.4                                                                        0.1%    Aeration:       1.0 l/min.                                 MgSO.sub.4.7H.sub.2 O                                                                    0.05%   Agitation:      430 rpm                                    Yeast Extract                                                                            0.2%    pH = 5.8 con-                                              Antifoam  10.0 g   trolled with 25% NaOH                                      d-H.sub.2 O                                                                              8.5 l   Duration:       4 days                                     ______________________________________                                    

SUBSTRATE PREPARATION

500 Grams of sclareol, 250 grams TWEEN® 80 and 1125 grams of water wereplaced in a blender and mixed for 5 minutes to form an emulsion.

Fermenter containing above medium was sterilized at 121° C. for 30minutes and cooled to 25° C. This fermenter was inoculated with 300 mlof 24 hour grown cells of Cryptococcus albidus (Saito [Skinner var.albidus]), ATCC 20918. 600 Grams of sclareol emulsion were added at timeof inoculation and 600 g portions of emulsion were added at 24, 48, and72 hours. At 96 hours, the contents of the fermenter were filteredthrough a 400 mesh sieve. The resulting crude solid product wasdissolved in IPA, filtered and the solution concentrated to crystallize;430 g of pure sclareolide were obtained.

FIG. 1 is a GC-MS spectrum for the initial reaction mass in this ExampleIII. The peak indicated by reference numeral 21 is the peak for thesclareol which is a mixture (80:20) of the compounds having thestructures: ##STR84## The peak indicated by reference numeral 20 is thepeak for the internal standard, the compound having the structure:##STR85##

FIG. 3 is the NMR spectrum for the sclareolide produced according tothis Example III having the structure: ##STR86##

EXAMPLE IV Production of Sclareolide From Sclareol Using Cryptococcusalbidus, ATCC 20918

Same medium and parameters were used in Example III. The mode ofsubstrate preparation and addition were changed.

One hundred sixty grams of sclareol powder and 80 g of TWEEN® 80 wereadded to the medium prior to sterilization.

Additional substrate was prepared by mixing finely ground sclareol (2parts) and TWEEN® 80 (1 part) to form a paste. Two hundred twenty fivegram portions of this paste were added at 24, 48, and 72 hours afterinoculation.

A total of 655 g of crude sclareolide having a purity of 67.34% wasobtained.

EXAMPLE V Preparation of Sclareolide From Sclareol Using Cryptococcusalbidus, ATCC 20921

The same medium and parameters were used as in Example IV. The amount ofsubstrate and agitation were changed.

One hundred fifty grams of sclareol and 75 grams of TWEEN® 80 were addedto the medium prior to sterilization.

Only 241 grams of substrate in paste form were added 24 hours afterinoculation. Agitation was started at 430 rpm and later increased to 630rpm.

A total of 491 g crude sclareol having a purity of 44% was obtained, a94.7% mole/mole conversion.

EXAMPLE VI Preparation of Sclareolide From Sclareol, Diol Intermediateand Diol Acetate Reactions ##STR87## The following medium was prepared:

    ______________________________________                                               NH.sub.4 NO.sub.3                                                                            0.2%                                                           KH.sub.2 PO.sub.4                                                                            0.1%                                                           MgSO.sub.4.7H.sub.2 O                                                                        0.05%                                                          Yeast Extract  0.2%                                                           Dextrose       0.5%                                                    ______________________________________                                    

A 10 liter fermenter was used with the following operating conditions:

    ______________________________________                                        Temperature:      25° C.                                               pH:               6.0                                                         Agitation:        430 rpm                                                     Sterilization:    121° C. for 30 mins.                                 ______________________________________                                    

The fermenter was inoculated with 100 ml of a 48 hour shake flaskculture grown on the same medium at 25° C. and 150 rpm.

After 24 hours growth, 200 ml aliquots were removed from the fermenterand centrifuged at 10,000 rpm for 10 minutes in a refrigeratedcentrifuge. The cells in each tube were washed twice with Butterfield'sbuffered phosphate.

BUFFER PREPARATION

Stock Solution:

    ______________________________________                                        Monopotassium hydrogen phosphate                                                                        34.0 g                                              Distilled water          500.0 ml                                             ______________________________________                                    

Adjust to pH 7.2 with about 175 ml 1.0 N sodium hydroxide solution;dilute to one liter and store.

Diluent:

Dilute 1.25 ml stock solution to 1 liter with distilled water. Preparedilution blanks in suitable containers. Sterilize at 121° C. for 15minutes.

The cells were then taken up in 100 ml of this buffer. The pH wasadjusted to 6 and then the material was transferred to a 500 ml flask.

Compounds tested:

a. Sclareol paste in TWEEN® 80 (2:1)=Compound 1.

b. Acetate, compound having the structure: ##STR88## mix in TWEEN® 80(1:1)=Compound 2. c. Diol paste, compound having the structure:##STR89## in TWEEN® 80 (1:1)=Compound 3.

300 ml Flasks containing 100 ml of buffer and cells (resting cells) wereplaced in shaker incubator at 25° C. and 150 rpm and samples wereanalyzed using TLC at 24, 48, and 72 hours against the standard knowncompound. In the following table:

P=Product, sclareolide having the structure: ##STR90## S=Substrate, oneof the compounds having the structure: ##STR91## I=Intermediate,compound having the structure: ##STR92## T=Trace.

                  TABLE II                                                        ______________________________________                                        Flask               Duration                                                  No.    Substrate    24 Hours   48 Hours                                                                             72 Hours                                ______________________________________                                        1      1 g of Compound                                                                            P + S + I  P + I  P + I                                          (1) added                                                              2      1 g of Compound                                                                            P + TS + I P + I  P + TI                                         (2) added                                                              3      1 g of Compound                                                                            TP + S     P + S  P + TS                                         (3) added                                                              ______________________________________                                    

EXAMPLE VII Formation of Diol Intermediate From Sclareol UsingBensingtonia ciliata, ATCC 20919

An experiment using the same procedure as Example II was carried outwith the following specifications:

    ______________________________________                                        Organism: Bensingtonia celiata, ATCC 20919 (IFF-8268C)                        MEDIUM          FERMENTER PARAMETERS                                          ______________________________________                                        NH.sub.4 NO.sub.3                                                                         40.0 g  Temperature:    25° C.                             Yeast Extract                                                                             40.0 g  Aeration:       2 l/min.                                  KH.sub.2 PO.sub.4                                                                         20.0 g  Agitation:      300 rpm                                   MgSO.sub.4.7H.sub.2 O                                                                     10.0 g  pH = 6.0 con-                                             Sclareol   160.0 g  trolled with 25% NaOH                                     TWEEN ® 80                                                                            80.0 g  Duration:       15 days                                   d-H.sub.2 O                                                                               19.0 l                                                            ______________________________________                                    

Fermenter containing above medium was sterilized at 121° C. for 30minutes and cooled at 25° C. This fermenter was inoculated with 1 literof 48 hour grown culture of Bensingtonia ciliata, ATCC 20919. After 15days of incubation, substrate was converted to product. The contents ofthe fermenter were filtered through a 400 mesh sieve. The resultingcrude solid product was air dried. Total of 101.7 g product wasobtained.

EXAMPLE VIII Preparation of Decahydro-3A, 6,6,9A-TetramethylNaphtho[2-1-Furan Reactions ##STR93##

Into a 3 neck 1 liter reaction vessel equipped with mechanical stirrer,thermometer and additional funnel and reflux condenser is added 124 mlVITRIDE® registered trademark of Eastman Kodak Company defining sodiumbis (2-methoxyethoxy) aluminum hydride.

To the VITRIDE® is added with stirring 500 ml toluene. 50 Grams ofsclareolide having the structure: ##STR94## prepared according to theprocedure of Example VI is dissolved in 120 ml toluene. Thesclareol/toluene solution is then added via addition funnel dropwiseover a period of 0.5 hours to the reaction mass and the temperature ofthe reaction mass is permitted to rise to 55° C.

The reaction mass is then heated to 80° C. for a period of two hours.

The reaction mass is then worked up by adding thereto 600 ml of 5%aqueous sodium hydroxide slowly while cooling with ice.

The toluene layer is removed and washed with saturated aqueous sodiumchloride followed by methyl alcohol and a sodium bicarbonate saturatedsolution (equal volumes).

The toluene layer is then concentrated yielding a solid weighing 43.34grams (86% yield).

The resulting solid has the structure: ##STR95##

1.5 Grams of the resulting compound is admixed with a solution of 12.0grams of potassium hydroxide in 30 ml water. The resulting mixture isplaced in a flask equipped with heater, stirrer and reflux condenser andrefluxed at 80° C. for a period of three hours. At the end of the threehour period, the resulting product is fractionally distilled at a vaportemperature of 162°-164° C. and a vacuum of 15 mm/Hg. yieldingsubstantially pure compound having the structure: ##STR96##

EXAMPLE IX Preparation of Cyclic Ether Reactions ##STR97##

The following medium was prepared:

    ______________________________________                                               NH.sub.4 NO.sub.3                                                                            0.2%                                                           KH.sub.2 PO.sub.4                                                                            0.1%                                                           MgSO.sub.4.7H.sub.2 O                                                                        0.05%                                                          Yeast Extract  0.2%                                                           Dextrose       1.0%                                                    ______________________________________                                    

Into a 500 ml flask was placed 100 ml medium and 1.0 g of a 50:50mixture of sclareol powder:TWEEN200 80. The flask was inoculated with400 microliters of isolate of Cryptococcus laurentii, ATCC 20920. Afterone week at 25° C. and 150 rpm (pH=6), the resulting product wasextracted with 330 ml ethyl acetate and the extract dried over anhydroussodium sulfate. The solvent was removed on a rotary evaporator.

The residue was dissolved in hot hexane and ethyl acetate. The resultingextract was permitted to evaporate for a period of 24 hours whereupon anoil was collected having the structure: ##STR98##

FIG. 4 is the NMR spectrum of the compound having the structure:##STR99##

What is claimed is:
 1. A process for producing a cylic ether having thestructure:which comprises cultivating a culture of the microorganism,Cryptococcus laurentii, ATCC 20920 to product said cyclic ether in arecoverable quantity upon the transformation of a substrate comprising acarbon source and, in addition, at least one compound selected from thegroup consisting of: ##STR100## and recovering said cyclic ether.
 2. Aprocess for producing a cyclic ether having the structure: ##STR101##which comprises cultivating the microorganism Cryptococcus laurentii,ATCC 20920 to produce said cylic ether in a recoverable quantity uponthe transformation of a substrate comprising a carbon source and, inaddition, at least one compound selected from the group consisting of:##STR102## under aerobic conditions in an aqueous nutrient mediumwherein: (i) the pH of the reaction mass is between about 2.5 and about9.0;(ii) the temperature of the reaction mass is between about 12° C.and about 33° C.; and (iii) the substrate concentration is between about0.1 grams per liter and about 130 grams per literand recovering saidcylic ether.
 3. The process of claim 2 wherein the carbon source isdextrose.
 4. The process of claim 2 wherein the substrate concentrationis between about 0.5 grams per liter and about 120 grams per liter.